Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

The HsdS polypeptide of the Type IC restriction enzyme Eco R124 is a sequence-specific DNA-binding protein

The HsdS and HsdM polypeptides of the type IC restriction enzyme EcoR124 have been purified independently and used in a set of gel retardation experiments to determine the minimum requirements for sequence-specific recognition of DNA by this enzyme. The HsdS polypeptide alone is able to bind to DNA in a sequence-specific manner. In addition, whilst the presence of the HsdM polypeptide gives rise to a stimulation of DNA binding by the HsdS subunit it is not clear whether, under the conditions of the experiments reported here, the HsdS subunit maintains the same interactions with the HsdM subunits observed in the absence of DNA.     

Nitrosating activity in Escherichia coli

Nitrosation activity was measured in Escherichia coli isolates and a range of nitrite reductase (nir) mutants. Activity was only detected in intact cells and could be inhibited by a number of treatments such as sonication and osmotic shock. Aerobically-grown cells had highest nitrosation activity compared to oxygen-limited ones. Inclusion of nitrite in growth media induced high activities of nitrite reductase and for some isolates, nitrosation. Analysis of nir mutants identified two which were unable to nitrosate. This result suggested that NADH-dependent nitrite reductase was implicated either directly or indirectly in nitrosation.     

A method for the fractionation of the high-mobility-group non-histone chromosomal proteins

A method is given for the preparation of four non-histone chromosomal proteins, one of which, protein 14, hitherto has not been isolated. The method also enables the preparation of histone H1 in gram quantities. The four non-histone chromosomal proteins so prepared are all polar molecules over 50% of each being composed of acidic and basic amino acids. It is also shown that protein 14 can be prepared from calf thymus without prior isolation of chromatin.     

Nitrogen dynamics in two antarctic streams

The many glacier meltwater streams of southern Victoria Land flow through catchments where life forms are almost entirely microbial. Allochthonous inputs of nitrogen from two study streams near McMurdo Sound were derived mostly from the melting glaciers (ca. 100–200 mg N m^–3) with some originating from N₂-fixation by heterocystous cyanobacteria (max. 939 mg N m^–2 year^–1). Thirty to fifty per cent of the glacier derived N was dissolved organic N and a major proportion of this was identified as urea N which was utilised by the rich algal and cyanobacterial mats in the streams. A nutrient budget for Fryxell Stream was estimated, quantifying uptake of urea-N and dissolved inorganic N and the release of dissolved organic (non urea) and particulate N by the stream communities. An index of in-stream nitrogen processing, the Net Uptake Length Constant in these streams was compared with that from temperate climates and was found to be similar. Despite the influence of low temperatures on microbial activity (mean daily water temperature = 5 °C) nutrient removal rates from these antarctic streams are high because of the large standing stock of microbial biomass there.     

Neuropathy Target Esterase: Rates of Turnover In Vivo Following Covalent Inhibition with Phenyl Di-n-Pentylphosphinate

Phenyl di-n-pentylphosphinate is a reasonably stable easily synthesized inhibitor of neuropathy target esterase (NTE) with low anticholinesterase activity. Like phenylmethylsulphonyl fluoride it protects hens against neuropathic effects of compounds such as diisopropylphosphorofluoridate. At intervals up to 15 days after dosing hens (10 mg/kg s.c. to inhibit 90% NTE) assays were made of catalytically active and of phosphinylated NTE in autopsy tissue. The sum of these components was always within the range of catalytic activity in undosed controls. However, the half-life of reappearance of active NTE was 2.07 days +/- 0.13 (SD, n = 6) for brain and 3.62 days +/- 0.23 (SD, n = 6) for spinal cord--shorter than after dosing with phenylmethylsulphonyl fluoride. It is proposed that: (1) The physiological turnover mechanism cannot distinguish between catalytically active and di-n-pentylphosphinylated NTE although initiation of organophosphate-induced delayed polyneuropathy might involve recognition of aged di-alkyl-phosphorylated NTE as "foreign". (2) The short half-lives indicate a slow spontaneous dephosphinylation of inhibited NTE occurs in vivo as well as de novo synthesis. The difference in half-lives for brain and spinal cord NTE may be due to different rates of synthesis de novo or (more likely) to different rates of spontaneous reactivation of the inhibited NTE in the two tissues.     

Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans

Summary The functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain. The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase). The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa. The QUTD gene shows strong homology with the N. crassa QA-Y gene and QUTG with the QA-X gene. QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5 non-coding regions show significant homology with UAS _GAL and UAS _QA sequences of the Saccharomyces cerevisiae and N. crassa Gal and QA systems. In addition, conserved 5 sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented.     

Opioid receptor dependent long-term potentiation: Peptidergic regulation of synaptic plasticity in the hippocampus

Rapid progress has been made towards understanding the synaptic physiology of excitatory amino acid transmission in the hippocampus. By comparison, the function of opioid peptides localized to some of the same pathways which use glutamate for fast excitation is poorly understood. Here I consider new evidence specifically implicating opioid peptides in long-term potentiation (LTP) induced by high-frequency stimulation of pathways which combine glutamate and opioid neurotransmission. This form of LTP is unique in that it depends on activation of opioid receptors, and unlike many excitatory systems in brain, it does not require activation of the (NMDA) type of glutamate receptor. Thus one of the main functions of opioids in the hippocampus may be to regulate activity-dependent changes in synaptic strength and neuronal excitability. At another level, “opioid” LTP may provide basic insights into peptidergic transmission and its functional interactions with classical neurotransmitters in the brain.     

Cytogenetic characterization of the L5178Y TK+/-3.7.2C mouse lymphoma cell line

The cytogenetic characterization of the L5178Y TK+/-3.7.2C mouse lymphoma cell line was carried out, utilizing G-banded metaphase chromosomes, to provide a karyotypic basis for the precise delineation of induced rearrangements in TK-/- mutants. Band-pattern measurements were used to construct ideograms which represent the position, number, size and staining intensity of the chromosome bands. The TK+/-3.7.2C cell line has been shown to provide quantitation of forward mutations induced at the autosomal thymidine kinase (TK) locus in this cell line. Chromosome analysis of the TK+/-3.7.2C cell line and derived TK-/- mutants has become important in demonstrating that the TK+/-----TK-/- assay may detect and distinguish between chromosomal events and smaller, perhaps point-mutation, events in mutant colonies.     

Clonal variations in keratin : Intermediate filament expression by human somatic cell hybrids

The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes.